Locking the two ends of tetrapeptidic HTLV-I protease inhibitors inside the enzyme

Adult T-cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy are only some of the more common end results of an infection with a human T-cell leukemia virus type 1 (HTLV-I). Expanding from our previous reports, we synthesized all different permutations of tetrapeptidic HTLV-I protease inhibitors using at least eight P(3)-cap and five P(1)(')-cap moieties. The inhibitors exhibited over 97% inhibition against HIV-1 protease and a wide range of inhibitory activity against HTLV-I protease.     

细丝蛋白A在细胞黏附和迁移中的作用

细胞迁移在发育、伤口愈合、炎症反应和肿瘤转移等多种病理生理过程中发挥重要作用。细丝蛋白A（filamin A,FlnA）是一种在各组织细胞中广泛表达的微丝结合蛋白,其表达异常导致细胞迁移功能障碍。该文回顾了相关的文献,首先介绍生理情况下细丝蛋白A的功能,接着介绍细丝蛋白A基因突变和表达异常导致的多种遗传性疾病及其与肿瘤转移的关系,突出细丝蛋白A对迁移的影响在这些疾病发病中的作用,最后深入探讨了细丝蛋白A影响细胞迁移和黏附的可能机制。     

Comparative processing and function of human and ferret cystic fibrosis transmembrane conductance regulator

The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is ΔF508, and this causes cystic fibrosis (CF). New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse ΔF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human ΔF508-CFTR. The processing behavior of ferret WT- and ΔF508-CFTR proteins remains unknown, and such information is important to predicting the utility of a ΔF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and ΔF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret ΔF508-CFTR processing was negligible, whereas low levels of processing of ΔF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not ΔF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTR(ΔF508/ΔF508) animal model may be useful.     

O-GlcNAcylation involvement in high glucose-induced cardiac hypertrophy via ERK1/2 and cyclin D2

Continuous hyperglycemia is considered to be the most significant pathogenesis of diabetic cardiomyopathy, which manifests as cardiac hypertrophy and subsequent heart failure. O-GlcNAcylation has attracted attention as a post-translational protein modification in the past decade. The role of O-GlcNAcylation in high glucose-induced cardiomyocyte hypertrophy remains unclear. We studied the effect of O-GlcNAcylation on neonatal rat cardiomyocytes that were exposed to high glucose and myocardium in diabetic rats induced by streptozocin. High glucose (30 mM) incubation induced a greater than twofold increase in cell size and increased hypertrophy marker gene expression accompanied by elevated O-GlcNAcylation protein levels. High glucose increased ERK1/2 but not p38 MAPK or JNK activity, and cyclin D2 expression was also increased. PUGNAc, an inhibitor of β-N-acetylglucosaminidase, enhanced O-GlcNAcylation and imitated the effects of high glucose. OGT siRNA and ERK1/2 inhibition with PD98059 treatment blunted the hypertrophic response and cyclin D2 upregulation. OGT inhibition also prevented ERK1/2 activation. We also observed concentric hypertrophy and similar changes of O-GlcNAcylation level, ERK1/2 activation and cyclin D2 expression in myocardium of diabetic rats induced by streptozocin. In conclusion, O-GlcNAcylation plays a role in high glucose-induced cardiac hypertrophy via ERK1/2 and cyclin D2.     

Control of hepatic nuclear superoxide production by glucose 6-phosphate dehydrogenase and NADPH oxidase-4

Redox-regulated signal transduction is coordinated by spatially controlled production of reactive oxygen species within subcellular compartments. The nucleus has long been known to produce superoxide (O(2)(·-)); however, the mechanisms that control this function remain largely unknown. We have characterized molecular features of a nuclear superoxide-producing system in the mouse liver. Using electron paramagnetic resonance, we investigated whether several NADPH oxidases (NOX1, 2, and 4) and known activators of NOX (Rac1, Rac2, p22(phox), and p47(phox)) contribute to nuclear O(2)(·-) production in isolated hepatic nuclei. Our findings demonstrate that NOX4 most significantly contributes to hepatic nuclear O(2)(·-) production that utilizes NADPH as an electron donor. Although NOX4 protein immunolocalized to both nuclear membranes and intranuclear inclusions, fluorescent detection of NADPH-dependent nuclear O(2)(·-) predominantly localized to the perinuclear space. Interestingly, NADP(+) and G6P also induced nuclear O(2)(·-) production, suggesting that intranuclear glucose-6-phosphate dehydrogenase (G6PD) can control NOX4 activity through nuclear NADPH production. Using G6PD mutant mice and G6PD shRNA, we confirmed that reductions in nuclear G6PD enzyme decrease the ability of hepatic nuclei to generate O(2)(·-) in response to NADP(+) and G6P. NOX4 and G6PD protein were also observed in overlapping microdomains within the nucleus. These findings provide new insights on the metabolic pathways for substrate regulation of nuclear O(2)(·-) production by NOX4.     

Synthesis and activity of tetrapeptidic HTLV-I protease inhibitors possessing different P3-cap moieties

The causative agent behind adult T-cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy is the human T-cell leukemia virus type 1 (HTLV-I). Tetrapeptidic HTLV-I protease inhibitors were designed on a previously reported potent inhibitor KNI-10516, with modifications at the P(3)-cap moieties. All the inhibitors showed high HIV-1 protease inhibitory activity (over 98% inhibition at 50nM) and most exhibited highly potent inhibition against HTLV-I protease (IC(50) values were less than 100nM).     

NaCl胁迫过程中喷施ALA对菘蓝幼苗生长及主要活性成分含量的影响

以来源于安徽亳州和山西运城的菘蓝（Isatis indigotica Fort.）幼苗为实验材料,对100 mmol·L-1NaCl胁迫条件下喷施50.0、25.0、16.7、12.5和0.0 mg·L-15-氨基乙酰丙酸（ALA）后幼苗部分生长指标、叶片中靛蓝和靛玉红含量以及根中表告依春含量的变化进行了研究。结果表明：在100 mmol·L-1NaCl单一胁迫条件下,来源于安徽亳州的幼苗单株叶鲜质量和叶片中靛蓝和靛玉红含量均低于对照,单株根鲜质量、根冠比以及根中表告依春含量均高于对照;来源于山西运城的幼苗单株叶和根鲜质量、叶片中靛玉红含量和根中表告依春含量均低于对照,根冠比和叶片中靛蓝含量均高于对照。在NaCl胁迫过程中喷施ALA对菘蓝幼苗生长和有效成分的积累均有不同效应。其中,喷施25.0 mg·L-1ALA后安徽亳州产幼苗单株叶和根鲜质量均最高且显著高于NaCl单一胁迫处理组;喷施16.7 mg·L-1ALA后山西运城产幼苗单株叶和根鲜质量均较高但与NaCl单一胁迫处理组无显著差异。喷施50.0 mg·L-1ALA后安徽亳州产幼苗叶片中靛蓝含量最高（0.376 mg·g-1）,喷施25.0 mg·L-1ALA后其叶片中靛玉红含量最高（9.977 mg·g-1）,喷施16.7 mg·L-1ALA后其根中表告依春含量最高（0.229 mg·g-1）,且均显著高于NaCl单一胁迫处理组;喷施16.7 mg·L-1ALA后山西运城产幼苗叶片中靛蓝含量最高（0.282 mg·g-1）,喷施12.5 mg·L-1ALA后其叶片中靛玉红含量和根中表告依春含量均最高（分别为4.526和0.301 mg·g-1）,且均显著高于NaCl单一胁迫处理组。研究结果表明：喷施适宜浓度ALA能够有效减轻NaCl胁迫对菘蓝幼苗生长的影响、提高其体内药用活性成分的含量;总体上看,产自安徽亳州的菘蓝幼苗的耐盐性较强,且不同种源适宜的ALA浓度也有一定差异。     

The late addition of core lipids to nascent apolipoprotein B100, resulting in the assembly and secretion of triglyceride-rich lipoproteins, is independent of both microsomal triglyceride transfer protein activity and new triglyceride synthesis.

Although microsomal triglyceride transfer protein (MTP) and newly synthesized triglyceride (TG) are critical for co-translational targeting of apolipoprotein B (apoB100) to lipoprotein assembly in hepatoma cell lines, their roles in the later stages of lipoprotein assembly remain unclear. Using N-acetyl-Leu-Leu-norleucinal to prevent proteasomal degradation, HepG2 cells were radiolabeled and chased for 0-90 min (chase I). The medium was changed and cells chased for another 150 min (chase II) in the absence (control) or presence of Pfizer MTP inhibitor CP-10447 (CP). As chase I was extended, inhibition of apoB100 secretion by CP during chase II decreased from 75.9% to only 15% of control (no CP during chase II). Additional studies were conducted in which chase I was either 0 or 90 min, and chase II was in the presence of [(3)H]glycerol and either BSA (control), CP (inhibits both MTP activity and TG synthesis),BMS-1976360-1) (BMS) (inhibits only MTP activity), or triacsin C (TC) (inhibits only TG synthesis). When chase I was 0 min, CP, BMS, and TC reduced apoB100 secretion during chase II by 75.3, 73.9, and 53.9%. However, when chase I was 90 min, those agents reduced apoB100 secretion during chase II by only 16.0, 19.2, and 13.9%. Of note, all three inhibited secretion of newly synthesized TG during chase II by 80, 80, and 40%, whether chase I was 0 or 90 min. In both HepG2 cells and McA-RH7777 cells, if chase I was at least 60 min, inhibition of TG synthesis and/or MTP activity did not affect the density of secreted apoB100-lipoproteins under basal conditions. Oleic acid increased secretion of TG-enriched apoB100-lipoproteins similarly in the absence or presence of either of CP, BMS, or TC. We conclude that neither MTP nor newly synthesized TG is necessary for the later stages of apoB100-lipoprotein assembly and secretion in either HepG2 or McA-RH7777 cells.